Algal desaturases

ABSTRACT

Provided herein are exemplary isolated nucleotide sequences encoding polypeptides having desaturase activity, which utilize fatty acids as substrates.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 13/458,914 filed on Apr. 27, 2012, titled “Algal Desaturases,”which claims the benefit and priority of U.S. Provisional PatentApplication Ser. No. 61/480,353 filed on Apr. 28, 2011, titled“Desaturases,” both of which are hereby incorporated by reference.

The present application is related to U.S. Non-Provisional patentapplication Ser. No. 12/581,812 filed on Oct. 19, 2009, titled“Homologous Recombination in an Algal Nuclear Genome,” which is herebyincorporated by reference.

The present application is related to U.S. Non-Provisional patentapplication Ser. No. 12/480,635 filed on Jun. 8, 2009, titled “VCP-BasedVectors for Algal Cell Transformation,” which is hereby incorporated byreference.

The present application is related to U.S. Non-Provisional patentapplication Ser. No. 12/480,611 filed on Jun. 8, 2009, titled“Transformation of Algal Cells,” which is hereby incorporated byreference.

REFERENCE TO SEQUENCE LISTINGS

The present application is filed with sequence listing(s) attachedhereto and incorporated by reference.

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to molecular biology, and more specifically, toalgal desaturases.

SUMMARY OF THE INVENTION

Isolated nucleotide sequences encoding polypeptides having desaturaseactivity, which utilize fatty acids as substrates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table of exemplary desaturases.

FIG. 2 illustrates the nucleotide sequence encoding desaturase 3 (SEQ IDNO:1).

FIG. 3 illustrates the nucleotide sequence encoding desaturase 5 (SEQ IDNO:2).

FIG. 4 illustrates the nucleotide sequence encoding desaturase 7 (SEQ IDNO:3).

FIG. 5 illustrates the nucleotide sequence encoding desaturase 9 (SEQ IDNO:4).

FIG. 6 illustrates the nucleotide sequence encoding desaturase 10 (SEQID NO:5).

FIG. 7 illustrates the amino acid sequence encoded by desaturase 3 (SEQID NO:6).

FIG. 8 illustrates the amino acid sequence encoded by desaturase 5 (SEQID NO:7).

FIG. 9 illustrates the amino acid sequence encoded by desaturase 7 (SEQID NO:8).

FIG. 10 illustrates the amino acid sequence encoded by desaturase 9 (SEQID NO:9).

FIG. 11 illustrates the amino acid sequence encoded by desaturase 10(SEQ ID NO:10).

DETAILED DESCRIPTION OF THE INVENTION

A fatty acid is a carboxylic acid with a long aliphatic tail (chain),which is either saturated or unsaturated. Saturated fatty acids arelong-chain carboxylic acids that usually have between 12 and 24 carbonatoms and have no double bonds. Unsaturated fatty acids have one or moredouble bonds between carbon atoms. Most naturally occurring fatty acidshave a chain of an even number of carbon atoms, from 4 to 28. A fattyacid desaturase is an enzyme that removes two hydrogen atoms from afatty acid, creating a carbon/carbon double bond. These desaturases areclassified as either Delta desaturases, indicating that the double bondis created at a fixed position from the carboxyl group of a fatty acid(for example, a delta nine (“Δ9”) desaturase creates a double bond atthe 9th position from the carboxyl end) or classified as Omegadesaturases (for example, an omega three (“ω3”) desaturase, whichcreates the double bond between the third and fourth carbon from themethyl end of the fatty acid).

Provided herein are isolated nucleotide sequences encoding polypeptideshaving desaturase activity, which utilize fatty acids as substrates.

The inventors sequenced the entire genome of algal genus Nannochloropsisand identified genes involved in fatty acid metabolism. They identifiedvarious desaturases, including exemplary desaturases which theydesignated as desaturase 3 (“desat3”), desaturase 5 (“desat5”),desaturase 7 (“desat7”), desaturase 9 (“desat9”), and desaturase 10(“desat10”).

The inventors manipulated the activities of the above-specifiedexemplary desaturase genes by:

1. Overexpression of the subject desaturase gene with a strong promoter.

2. Promoter replacement or promoter insertion in front of the subjectdesaturase gene within the genome via homologous recombination.

3. Knock out of the subject desaturase gene via insertion of atransformation construct into the gene or replacement of a part of orthe entire subject desaturase gene via homologous recombination.

Exemplary support for the above-mentioned methods may be found in U.S.Non-Provisional patent application Ser. No. 12/581,812 filed on Oct. 19,2009, titled “Homologous Recombination in an Algal Nuclear Genome,” U.S.Non-Provisional patent application Ser. No. 12/480,635 filed on Jun. 8,2009, titled “VCP-Based Vectors for Algal Cell Transformation,” and U.S.Non-Provisional patent application Ser. No. 12/480,611 filed on Jun. 8,2009, titled “Transformation of Algal Cells,” all of which are herebyincorporated by reference.

Accordingly, the inventors were able to manipulate the activities of thevarious exemplary desaturases for the purpose of modifying the contentsof certain fatty acids within algal genus Nannochloropsis.

FIG. 1 is a table of exemplary desaturases. The table includes thenumber of the respective desaturase, captioned “Desaturase No.”, thecorresponding sequence identifier number, captioned “SEQ ID NO”, thetype of desaturase, captioned “Desaturase Type”, the relative increaseor decrease in desaturase gene regulation with respect to the presenceor absence of Nitrogen, captioned “Times Up/Down Regulated in thePresence/Absence of Nitrogen”, a first desaturase substrate, captioned“Substrate 1”, the resulting first product, captioned “Product 1”, asecond desaturase substrate (if applicable), captioned “Substrate 2”,the resulting second product (if applicable), captioned “Product 2”,whether the desaturase gene was up-regulated or down-regulated in aparticular mutant construct (with respect to the Substrate 1 to Product1 reaction), captioned “Gene Regulation in Mutant Up/Down”, the observedincrease or decrease of the corresponding substrate to product reactionby a mutant construct (as compared to a wild-type Nannochloropsiscontrol cell), captioned “Substrate 1 to Product 1 Conversion” whetherthe desaturase gene was up-regulated or down-regulated in a particularmutant construct (with respect to the Substrate 2 to Product 2reaction)(if applicable), captioned “Gene Regulation in Mutant Up/Down”and the observed increase or decrease of the corresponding substrate toproduct reaction by a mutant construct (as compared to a wild-typeNannochloropsis control cell), captioned “Substrate 2 to Product 2Conversion” (if applicable).

As shown in FIG. 1, the inventors identified five exemplary desaturases,designated as desaturase 3 (“desat3”), desaturase 5 (“desat5”),desaturase 7 (“desat7”), desaturase 9 (“desat9”), and desaturase 10(“desat10”). Each desaturase has a corresponding SEQ ID NO, reflecting anucleotide sequence for the respective desaturase. The desaturase type(which indicates corresponding enzymatic activity) is also shown inFIG. 1. The column captioned “Times Up/Down Regulated in thePresence/Absence of Nitrogen” reflects whole transcriptome comparisonsof samples grown under Nitrogen sufficient (“N+”) or Nitrogen deficientconditions (“N-”). The columns captioned “Substrate 1 to Product 1Conversion” and “Substrate 2 to Product 2 Conversion” (if applicable)show the observed increase or decrease of the corresponding substrate toproduct reactions by a mutant construct (as compared to a wild-typeNannochloropsis control cell).

Referring again to the table in FIG. 1, desaturase 3, a Delta 12desaturase, is approximately 1.5 times up-regulated in the absence ofNitrogen. Desaturase 3 increases the conversion of Palmitolenic acid(16:2(n-7)) to 6,9,12-hexadecatrienoic acid (16:3(n-4)) by approximately85% (when compared to a wild-type Nannochloropsis control cell).Desaturase 3 also increases the conversion of Oleic acid,cis-9-octadecenoic acid (18:1(n-9)) to Linoleic acid, all-cis-9,12-octadecadienoic acid (18:2(n-6)) by approximately 50% (when comparedto a wild-type Nannochloropsis control cell).

Desaturase 5, as shown in the table in FIG. 1, is also a Delta 12desaturase. Desaturase 5 is not regulated in the presence or absence ofNitrogen. Desaturase 5 decreases the conversion of Oleic acid,cis-9-octadecenoic acid (18:1(n-9)) to 12-octadecadienoic acid(18:2(n-6)) by approximately 100% (when compared to a wild-typeNannochloropsis control cell).

Desaturase 7, shown in the table in FIG. 1, is an Omega 3 desaturase.Desaturase 7 is approximately 4.0 times up-regulated in the presence ofNitrogen. Desaturase 7 decreases the conversion of Arachidonic acid,all-cis-5,8,11,14-eicosatetraenoic acid (20:4(n-6))(“ARA”) toEicosapentaenoic acid, all-cis-5,8,11,14,17-eicosapentaenoic acid(20:5(n-3))(“EPA”) by approximately 60% (when compared to a wild-typeNannochloropsis control cell). In fact, the EPA to ARA ratio changesfrom about 7.5 to about 22, an approximate 200% increase. Desaturase 7increases the conversion of Linoleic acid, all-cis-9, 12-octadecadienoicacid (18:2(n-6)) to α-Linolenic acid, all-cis-9,12,15-octadecatrienoicacid (18:3(n-3)) by approximately 650% (when compared to a wild-typeNannochloropsis control cell). In fact, the ratio of α-Linolenic acid,all-cis-9,12,15-octadecatrienoic acid (18:3(n-3)) to Linoleic acid,all-cis-9, 12-octadecadienoic acid (18:2(n-6)) changes fromapproximately 2.5 to approximately 15.8, an approximate 550% increase.

Desaturase 9, shown in the table in FIG. 1, is a Delta 9 desaturase.Desaturase 9 is approximately 6.6 times up-regulated in the absence ofNitrogen. Desaturase 9 increases the conversion of Palmitic acid, orhexadecanoic acid (16:0) to Palmitoleic acid, cis-9-hexadecenoic acid(16:1(n-7)) by approximately 25%, while increasing the ratio ofPalmitoleic acid, cis-9-hexadecenoic acid (16:1(n-7)) to Palmitic acid,or hexadecanoic acid (16:0) from about 0.48 to 0.6, an approximateincrease of 25%.

Desaturase 10, shown in the table in FIG. 1, is a Delta 9 desaturase.Desaturase 9 is approximately 5.0 times up-regulated in the absence ofNitrogen. Desaturase 9 increases the conversion of Palmitic acid, orhexadecanoic acid (16:0) to Palmitoleic acid, cis-9-hexadecenoic acid(16:1(n-7)) by approximately 34%, while increasing the ratio ofPalmitoleic acid, cis-9-hexadecenoic acid (16:1(n-7)) to Palmitic acid,or hexadecanoic acid (16:0) from about 1.02 to 1.19, an approximateincrease of 16%.

FIG. 2 illustrates the nucleotide sequence encoding desaturase 3 (SEQ IDNO:1).

The inventors found that desaturase 3 is slightly up-regulated underNitrogen starvation. The inventors prepared a construct utilizingdesaturase 3 that included a strong upstream promoter. This resulted inexpression of elevated amounts of 16:3n4 and 18:2n6 fatty acids.

FIG. 3 illustrates the nucleotide sequence encoding desaturase 5 (SEQ IDNO:2).

The inventors found that desaturase 5 encodes a fatty acid desaturasewith high homology to Delta 12 desaturases. The inventors also foundthat overexpression of this desaturase gene under the control of aninducible Urease promoter leads to higher expression levels of 18:1n9fatty acids and poly unsaturated fatty acids (“PUFAs”) with 18 or morecarbon atoms, when the constructs are grown under Nitrogen starvationconditions (please note: the promoter is induced under Nitrogenstarvation).

In other experiments, the inventors determined that the desaturationstep at the Delta 12 position is likely a major bottleneck forchanneling carbon into the PUFA biosynthesis pathway. While 18:1n9 fattyacids are steadily increasing during Nitrogen starvation, 18:2n6 fattyacids (derived from the Delta 12 desaturation of said 18:1n9 fattyacids) are decreasing, as are all fatty acids in the pathway leading tothe production of Eicosapentaenoic acid (“EPA”). The inventors concludedthat the desaturase 5 gene increases carbon flux into the PUFAbiosynthesis pathway during Nitrogen starvation if the desaturase 5 geneis over-expressed.

FIG. 4 illustrates the nucleotide sequence encoding desaturase 7 (SEQ IDNO:3).

The inventors prepared various constructs (promoter replacements,knock-outs, and over expression constructs) and found thatdown-regulation of the desaturase 7 gene results in a lowerEPA/Arachidonic acid (“ARA”) ratio, i.e. less ARA is desaturated to EPA.The inventors observed in mutant constructs that lower desaturase 7transcription is due to an exchange of the native promoter in thewild-type Nannochloropsis cells. The inventors observed that the mutantconstructs had nearly double the levels of ARA with less levels of EPA,when compared to the wild-type Nannochloropsis control cells.

The inventors also observed that up-regulation of the desaturase 7 generesults in higher 18:3n3/18:2n6 and EPA/ARA ratios, i.e. more 18:2n6 isconverted to 18:3n3, and more ARA is converted into EPA. Accordingly,the inventors observed that the EPA/ARA ratio was nearly doubled.

FIG. 5 illustrates the nucleotide sequence encoding desaturase 9 (SEQ IDNO:4).

FIG. 6 illustrates the nucleotide sequence encoding desaturase 10 (SEQID NO:5).

The inventors observed that both desaturase 9 and desaturase 10 appearto be Delta 9 desaturases acting primarily on 16:0 fatty acids or on16:0 fatty acids attached to other compounds. Promotor exchange studies,in which the inventors exchanged the native wild-type promoter ofNannochloropsis against a strong promoter, revealed an up-regulation ofsaid activity under Nitrogen deficient conditions. Thus, under Nitrogenstarvation, a high percentage of fatty acids are channeled to theaccumulation of 16:1n7 fatty acids through the action of the desaturase9 gene, meaning less fatty acids are entering the PUFA pathway. Theinventors also replaced the promoters of the desaturase 9 genes withpromoters of moderate strength and which are putatively not regulatedwhen cells enter Nitrogen starvation, with the goal to avoid carbon fluxinto the biosynthesis of 16:1n7 and 18:1n7 fatty acids and to increasecarbon flux into the PUFA biosynthesis pathway during starvation. Theinventors found that these genes are excellent targets forover-expression, in order to achieve elevated PUFA biosynthesis.Down-regulation of these (or other) genes, as an example, by replacementof the endogenous promoter or insertion of a weaker promoter in front ofthe respective desaturase gene may lead to a higher content of shortchain fatty acids. Down-regulation of transcription could also beachieved, in some cases, by insertion of a commonly strong promoter infront of the respective desaturase gene, presumably by modifying therespective chromatin arrangement around the desaturase gene, thusleading to a lower transcription level. Also, the introduction of pointmutations into the desaturase gene when inserting another promoter infront of the desaturase gene via the homologous recombination flanks maylead to an altered activity of the respective gene products.

The inventors also observed an increase of 16:1n7 lipids in a selecteddesaturase 10 over expression mutant, clearly demonstrating that thereis an approximately 40% increase in 16:1n7 fatty acids in this mutantwhen compared to the wild-type Nannochloropsis cells during the sameexperiment.

FIG. 7 illustrates the amino acid sequence encoded by desaturase 3 (SEQID NO:6).

FIG. 8 illustrates the amino acid sequence encoded by desaturase 5 (SEQID NO:7).

FIG. 9 illustrates the amino acid sequence encoded by desaturase 7 (SEQID NO:8).

FIG. 10 illustrates the amino acid sequence encoded by desaturase 9 (SEQID NO:9).

FIG. 11 illustrates the amino acid sequence encoded by desaturase 10(SEQ ID NO:10).

While various embodiments have been described above, it should beunderstood that they have been presented by way of example only, and notlimitation. Thus, the breadth and scope of a preferred embodiment shouldnot be limited by any of the above-described exemplary embodiments.

What is claimed is:
 1. An isolated nucleotide sequence encoding apolypeptide having desaturase activity, the nucleotide sequence havingat least 95% sequence identity to SEQ ID NO:1.
 2. The isolatednucleotide sequence of claim 1 wherein the sequence encodes afunctionally active desaturase which utilizes a fatty acid as asubstrate.
 3. The isolated nucleotide sequence of claim 2, wherein thefunctionally active desaturase comprises amino acids having the sequenceset forth as SEQ ID NO:6 or a sequence at least 95% identical thereto.4. An isolated nucleotide sequence encoding a polypeptide havingdesaturase activity, the nucleotide sequence having at least 95%sequence identity to SEQ ID NO:2.
 5. The isolated nucleotide sequence ofclaim 4 wherein the sequence encodes a functionally active desaturasewhich utilizes a fatty acid as a substrate.
 6. The isolated nucleotidesequence of claim 5, wherein the functionally active desaturasecomprises amino acids having the sequence set forth as SEQ ID NO:7 or asequence at least 95% identical thereto.
 7. The isolated nucleotidesequence of claim 1 wherein the sequence is derived from algae.
 8. Theisolated nucleotide sequence of claim 7 wherein the algae is of genusNannochloropsis.